CN113884414A - Calibration counting plate and microscope lens calibration method - Google Patents
Calibration counting plate and microscope lens calibration method Download PDFInfo
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- CN113884414A CN113884414A CN202110986453.6A CN202110986453A CN113884414A CN 113884414 A CN113884414 A CN 113884414A CN 202110986453 A CN202110986453 A CN 202110986453A CN 113884414 A CN113884414 A CN 113884414A
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- 238000000034 method Methods 0.000 title claims abstract description 24
- 210000004027 cell Anatomy 0.000 claims description 30
- 210000001772 blood platelet Anatomy 0.000 claims description 4
- 210000003743 erythrocyte Anatomy 0.000 claims description 4
- 210000000265 leukocyte Anatomy 0.000 claims description 4
- 230000000007 visual effect Effects 0.000 claims description 4
- 239000003292 glue Substances 0.000 claims 1
- 210000004369 blood Anatomy 0.000 abstract description 16
- 239000008280 blood Substances 0.000 abstract description 16
- 230000006872 improvement Effects 0.000 description 7
- 230000008569 process Effects 0.000 description 4
- 239000002390 adhesive tape Substances 0.000 description 3
- 238000013459 approach Methods 0.000 description 3
- 238000004820 blood count Methods 0.000 description 2
- 239000000306 component Substances 0.000 description 2
- 238000010329 laser etching Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 230000004075 alteration Effects 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 239000012503 blood component Substances 0.000 description 1
- 238000009534 blood test Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000010147 laser engraving Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000009666 routine test Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N1/31—Apparatus therefor
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/483—Physical analysis of biological material
- G01N33/487—Physical analysis of biological material of liquid biological material
- G01N33/49—Blood
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- G01N2015/012—
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N2015/1006—Investigating individual particles for cytology
Abstract
The invention discloses a calibration counting plate and a microscope lens calibration method, wherein the calibration counting plate comprises a bottom plate and an upper cover which are buckled together, at least one counting cell with an upward opening is formed between the bottom plate and the upper cover, and a calibration target used for performing calibration operation on a microscope lens is engraved in the region of the bottom plate corresponding to the counting cell. According to the calibration counting plate, the calibration target is scribed on the bottom wall of the counting cell and is used as an observed object, so that the depth of field reference position of a microscope lens in the moving direction can be found out, and the counting plates are manufactured in batches with uniform specifications, so that the calibration counting plate can represent the condition of counting plates in the same batch, namely the counting plates in the batch correspond to the same depth of field reference position, and during actual use, calibration can be carried out at any time before real observation, so that dependence on blood samples is avoided; and the position of the calibration target relative to the counting cell is fixed, so that the consistency of the calibration result can be ensured.
Description
Technical Field
The invention relates to a calibration counting plate and a microscope lens calibration method.
Background
In the field of routine blood tests, red blood cells, white blood cells, platelets and the like are observed and counted by using a microscope, so that information about the concentration and the like of blood components is obtained, and diagnosis of related diseases is further assisted.
In the above application scenario, since blood cells belong to microscopic objects, the range in which the lens can move up and down is small during observation, that is, the depth of field is small, generally 300 μm, and if the lens moves beyond the depth of field, an image cannot be obtained. After each microscope leaves a factory, the structural components of each microscope are not absolutely flat, and certain gaps exist between the components in physical connection, so when the counting plate is driven by the microscope to move horizontally, the counting plate also fluctuates up and down actually, and the phenomenon brings certain trouble to observation. The existing solution is to take a counting plate, drip blood sample and observe on a computer, and find the reference line of the lens in the height direction through adjustment, so as to set the depth of field range of the lens. The method can not get rid of the dependence on the blood sample, and can not calibrate the counting plates of a certain batch under the condition of no blood sample, so that the matched depth-of-field data of the counting plates of the batch can not be determined when the counting plates leave a factory.
In view of the above, a new technical solution is needed to solve the above technical problems.
Disclosure of Invention
The invention aims to provide a calibration counting plate and a microscope lens calibration method, which can enable the position of a calibration lens to get rid of dependence on blood samples, namely the reference position of the depth of field of the counting plate during on-machine observation can be determined without dripping the blood samples when the counting plate leaves a factory.
In order to achieve the purpose, the invention adopts the following technical means:
a calibration counting plate comprises a bottom plate and an upper cover which are buckled together, at least one counting cell with an upward opening is formed between the bottom plate and the upper cover, and a calibration target used for performing calibration operation on a microscope lens is engraved in an area of the bottom plate corresponding to the counting cell.
As a further improvement, the calibration target comprises a calibration groove line, a calibration concave point or a calibration grid.
As a further improvement, the calibration slot lines are arranged in a plurality of parallel, the width of each calibration slot line is 100 micrometers, the interval between every two calibration slot lines is 150 micrometers, and the depth of each calibration slot line is 5-10 micrometers.
As a further improvement, the bottom plate and the upper cover are bonded together through a double-sided adhesive tape, and a sample dripping hole is formed in the position, corresponding to the counting cell, of the upper cover.
As a further refinement, the calibration target is coated with a staining agent.
A method for calibrating the position of a microscope lens by using the calibration counting plate comprises the following steps:
horizontally placing the calibration counting plate on an object stage;
adjusting a lens to focus in the Z direction, so that the definition of a calibration target image in the visual field reaches a preset value;
when the image of the calibration target reaches a preset value, recording the current Z-axis position as A;
the working range of the lens on the Z axis is set as follows: and the range of 300 microns above and below the reference line A.
As a further improvement, the operating range of the lens in the Z axis is set as follows: the method comprises the following steps of taking A as a reference line and within the range of 300 micrometers from top to bottom: the working range of the lens on the Z axis is set as follows: between a-100 microns and a +200 microns.
As a further improvement, before the step of adjusting the lens to focus in the Z direction to make the sharpness of the calibrated target image in the field of view reach a preset value, the method further comprises:
the stage is moved horizontally to align the lens with the position of one of the cells.
As a further improvement, the counting plate is provided with three counting cells which respectively comprise a red blood cell counting cell, a white blood cell counting cell and a platelet counting cell.
As a further improvement, the microscope is an inverted microscope.
Compared with the prior art, the invention has the following technical effects:
according to the calibration counting plate, the calibration target is scribed on the bottom wall of the counting cell and is used as an observed object, so that the depth of field reference position of a microscope lens in the moving direction can be found out, and the counting plates are manufactured in batches with uniform specifications, so that the calibration counting plate can represent the condition of counting plates in the same batch, namely the counting plates in the batch correspond to the same depth of field reference position, and during actual use, calibration can be carried out at any time before real observation, so that dependence on blood samples is avoided; and the position of the calibration target relative to the counting cell is fixed, so that the consistency of the calibration result can be ensured.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a perspective view of a calibration counter plate according to a preferred embodiment of the present invention;
FIG. 2 is an exploded perspective view of a calibration counter plate according to a preferred embodiment of the present invention;
FIG. 3 is a cross-sectional view of a bottom plate of the calibration and counting plate according to a preferred embodiment of the present invention;
fig. 4 shows a flow chart of the microscope lens calibration method of the present invention.
Description of the main element symbols:
calibrating the counting plate 10; a base plate 11; an upper cover 12; a double-sided adhesive tape 13; a dripping hole 14; the slot line 15 is calibrated.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the accompanying drawings are illustrative only for the purpose of explaining the present invention, and are not to be construed as limiting the present invention.
In the description of the present invention, it is to be understood that the terms "central," "longitudinal," "lateral," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," "counterclockwise," "axial," "radial," "circumferential," and the like are used in the orientations and positional relationships indicated in the drawings for convenience in describing the invention and to simplify the description, and are not intended to indicate or imply that the referenced device or element must have a particular orientation, be constructed and operated in a particular orientation, and are not to be considered limiting of the invention.
Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the present invention, "a plurality" means two or more unless specifically defined otherwise.
In the present invention, unless otherwise expressly stated or limited, the terms "mounted," "connected," "secured," and the like are to be construed broadly and can, for example, be fixedly connected, detachably connected, or integrally formed; can be mechanically or electrically connected; either directly or indirectly through intervening media, either internally or in any other relationship. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.
In the present invention, unless otherwise expressly stated or limited, the first feature "on" or "under" the second feature may be directly contacting the first and second features or indirectly contacting the first and second features through an intermediate. Also, a first feature "on," "over," and "above" a second feature may be directly or diagonally above the second feature, or may simply indicate that the first feature is at a higher level than the second feature. A first feature being "under," "below," and "beneath" a second feature may be directly under or obliquely under the first feature, or may simply mean that the first feature is at a lesser elevation than the second feature.
Examples
The counting plate is applied to a cell counting observation scene, a counting cell is arranged on the counting plate, a blood sample is dripped into the counting cell, and then the counting cell and the blood sample are placed under a microscope together for observation, so that relevant information about blood concentration, such as a blood routine test in medicine, is obtained.
The embodiment discloses a calibration counting plate 10 and a method for calibrating a lens of a microscope by using the calibration counting plate 10.
Referring to fig. 1 to 3, the calibration counting plate 10 includes a bottom plate 11 and an upper cover 12 that are fastened together, at least one counting cell (not labeled in the figures) with an upward opening is formed between the bottom plate 11 and the upper cover 12, and a calibration target for calibrating a microscope lens is marked on a region of the bottom plate 11 corresponding to the counting cell.
According to the calibration counting plate 10, the calibration target is scribed on the bottom wall of the counting cell and is used as an observed object, so that the depth of field reference position of a microscope lens in the moving direction can be found out, and the counting plates are manufactured in batches in a unified specification, so that the calibration counting plate 10 can represent the condition of counting plates in the same batch, namely the counting plates in the batch correspond to the same depth of field reference position, and during actual use, calibration can be carried out at any time before real observation, and dependence on blood samples is avoided; and the position of the calibration target relative to the counting cell is fixed, so that the consistency of the calibration result can be ensured.
In one embodiment, the calibration counting plate 10 is provided with three counting cells for observing and counting red blood cells, white blood cells and platelets. The bottom plate 11 and the upper cover 12 are glued together through a double-sided adhesive tape 13, and a sample dripping hole 14 is formed in the position, corresponding to the counting cell, of the upper cover 12.
Further, in order to distinguish the three counting wells, the shapes of the dripping holes 14 of the three counting wells can be set to different geometric shapes, for example, two of the three counting wells are set to be square, and the other counting well is set to be circular; of course, other arrangements are possible.
Referring to fig. 3, in one embodiment, the calibration target is a calibration groove line 15, i.e., a groove line is formed on the upper surface of the bottom plate 11 by a laser etching or engraving process.
Preferably, the calibration slot lines 15 are provided in a plurality and parallel to each other, a width of each calibration slot line 15 is 100 micrometers, a distance between every two calibration slot lines 15 is 150 micrometers, and a depth of each calibration slot line 15 is 5-10 micrometers. The size is set to be appropriate in density, and the calibration slot line 15 can be quickly found out when the microscope is used for observation. The cross-section of the calibration slot line 15 may be semicircular, rectangular, prismatic or other shapes, which is not limited in the present invention.
Preferably, the calibration slot line 15 is coated with a coloring agent, such as red, brown and the like, so that the color contrast between the calibration slot line 15 and the surrounding environment is more obvious, and the operator can conveniently observe the calibration slot line.
Referring to fig. 4, in one embodiment, the method for calibrating a lens of a microscope by using the calibration counting plate 10 includes the following steps:
s100, horizontally placing the calibration counting plate 10 on an object stage.
An observation window is arranged on the objective table, and the calibration counting plate 10 is fixed at the position.
And S200, horizontally moving the object stage to enable the calibration counting plate 10 to move to a position where the lens is vertically aligned with one counting cell.
The lens of the microscope is fixed in position in the horizontal direction, so that the calibration and counting plate 10 needs to be moved to a proper position for observation. The microscope stage is provided with a mechanism which can drive the calibration and counting plate 10 to move horizontally, such as a sliding plate, a sliding rail, a screw rod and the like.
In one embodiment, since the number of the counting cells is three, for the counting plate, calibration work needs to be performed by selecting a point in each of the three counting cells.
S300, adjusting the lens to focus in the Z direction, and enabling the image definition of the calibration slot line 15 in the visual field to reach a preset value.
Specifically, in the process that the lens approaches the calibration counting plate 10 from a relatively far initial position, coarse adjustment is performed to enable the lens to move rapidly, and when the lens moves to a position close to the calibration counting plate 10, fine adjustment is performed to enable the lens to approach the calibration counting plate 10 slowly. The operator looks at the situation in the eyepiece at the same time: in the course of coarse adjustment, no image exists in the ocular lens; and as the lens approaches the calibration counting plate 10, an image of a calibration groove line 15 appears in the ocular lens, fine adjustment is performed, and in the process, the image definition is gradually increased.
It should be noted that the focusing adjustment of the lens in the Z direction in step S300 may be performed manually, or may be performed automatically by setting a program.
S400, when the image of the calibration target reaches a preset value, recording the current Z-axis position as A.
When the definition of the image in the ocular reaches the maximum value, representing that the focusing of the lens on the bottom wall of the counting cell is successful under the position, recording the current position of the Z axis, and taking the position as a datum line.
It should be noted that, whether the image reaches the preset definition value or not may be determined by visual observation or by an algorithm. The method for calculating and judging the definition of the image through the algorithm has more mature schemes in the prior art, and the invention is not repeated herein.
S500, setting the working range of the lens on the Z axis as follows: and the range of 300 microns above and below the reference line A.
Specifically, in one embodiment, the working range of the lens in the Z axis is set as follows: between a-100 microns and a +200 microns. The purpose of this setting is that after the blood sample is dropped into the counting chamber, it will be stacked on the bottom plate 11 to form a blood sample layer with a certain thickness, and the observation count of the blood sample needs to be spread over the whole blood sample layer, so the lens is set to a value above which is greater than that below.
The microscope of the present embodiment is an inverted microscope, i.e., the lens is below the stage.
In the above embodiments, the calibration target is a strip-shaped calibration groove line 15, and it should be understood that in some other embodiments, the calibration target may also be configured as a calibration concave point, a calibration grid, etc., as long as it is a structure capable of serving as a target in a lens calibration process.
It should be noted that the calibration groove lines 15, the calibration pits, or the calibration grids may be made by laser etching or by engraving with a graver on a machine tool, and the manufacturing process and tools of the target in the prior art are various, and the present invention is not limited to this, and will not be described herein again.
The microscope of the invention can also be provided with an input device and a storage device, for example, a touch screen is arranged, after one calibration operation is completed, the coordinate information of the position A can be recorded through the touch screen and stored in the storage device, and when the counting plate in the same batch as the calibration counting plate 10 is used subsequently, the calibration result can be directly called, thereby accelerating the efficiency.
It can be understood that the above-mentioned calibration counting plate 10 is provided with three counting cells, so that at least three reference values of the three points, which are respectively taken from a certain point of the corresponding areas of the three counting cells, need to be recorded for the calibration counting plate 10.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (10)
1. The calibration counting plate is characterized by comprising a bottom plate and an upper cover which are buckled together, wherein at least one counting cell with an upward opening is formed between the bottom plate and the upper cover, and a calibration target used for performing calibration operation on a microscope lens is engraved in a region of the bottom plate corresponding to the counting cell.
2. The calibration count plate of claim 1 wherein said calibration targets comprise calibration groove lines, calibration pits, or calibration grids.
3. The nominal count plate of claim 2, wherein said nominal groove lines are provided in a plurality and parallel relationship, each of said nominal groove lines having a width of 100 microns, each of said nominal groove lines being spaced 150 microns apart, each of said nominal groove lines having a depth of 5-10 microns.
4. The calibration counting plate of claim 1, wherein said bottom plate and said upper cover are glued together by double-sided glue, and said upper cover is provided with a sample dropping hole at a position corresponding to said counting chamber.
5. The calibrated count plate of claim 1, wherein said calibration targets are coated with a stain.
6. A method of calibrating a microscope lens using the calibration counter plate of any of claims 1-5, comprising the steps of:
horizontally placing the calibration counting plate on an object stage;
adjusting a lens to focus in the Z direction, so that the definition of a calibration target image in the visual field reaches a preset value;
when the image of the calibration target reaches a preset value, recording the current Z-axis position as A;
the working range of the lens on the Z axis is set as follows: and the range of 300 microns above and below the reference line A.
7. The method of claim 6, wherein the working range of the lens in the Z-axis is set to: the method comprises the following steps of taking A as a reference line and within the range of 300 micrometers from top to bottom: the working range of the lens on the Z axis is set as follows: between a-100 microns and a +200 microns.
8. The method of claim 6, wherein before the step of adjusting the lens to focus in the Z direction to make the sharpness of the target image in the field of view reach a predetermined value, the method further comprises:
the stage is moved horizontally to align the lens with the position of one of the cells.
9. The method of claim 6 wherein said counting plate is provided with three cells, including a red blood cell, a white blood cell and a platelet cell.
10. The method of claim 6, wherein the microscope is an inverted microscope.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202110986453.6A CN113884414A (en) | 2021-08-26 | 2021-08-26 | Calibration counting plate and microscope lens calibration method |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202110986453.6A CN113884414A (en) | 2021-08-26 | 2021-08-26 | Calibration counting plate and microscope lens calibration method |
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| CN213091449U (en) * | 2020-08-15 | 2021-04-30 | 湖南伊鸿健康科技有限公司 | Multifunctional counting plate and electron microscope |
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2021
- 2021-08-26 CN CN202110986453.6A patent/CN113884414A/en active Pending
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| JP2004012192A (en) * | 2002-06-04 | 2004-01-15 | Olympus Corp | Measuring microscope device, its display method, and its display program |
| CN201421323Y (en) * | 2009-03-31 | 2010-03-10 | 深圳市纳研科技有限公司 | Two-dimensional calibration board |
| CN103725604A (en) * | 2014-01-08 | 2014-04-16 | 爱威科技股份有限公司 | Cell counting plate |
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